Fatty acyl-CoA Research Articles

Abstract 1484: SCD-1 blockade sensitizes triple negative breast cancer to immune checkpoint inhibition

Abstract The increase in fatty acid metabolism is a hallmark of cancer. Stearoyl CoA desaturase (SCD) is an endoplasmic reticulum (ER) enzyme that catalyzes D9 cis désaturation of fatty acyl-CoA substrates. One of its isoforms, SCD1, is overexpressed in several cancers including breast cancer. The higher expression of SCD1 in breast cancer patients correlates with significantly shorter relapse-free and overall survival rates, indicating that SCD1 may be a therapeutic target. We have previously developed and published a novel SCD1 inhibitor, SSI4. Metabolic reprogramming facilitates tumor growth by promoting immune suppression in tumors. Fundamental metabolic processes like fatty acid (FA) metabolism are involved in T cell activation and differentiation and their modulation can differentially affect the development of T helper cell lineages. FA synthesis and oxidation are essential for the development and functioning of CD8+ T memory cells and are contributing factors that influence CD4+ T effector and Treg cell development. These associations between metabolism and immune cells lead us to our hypothesis that aberrant de novo lipogenesis is linked to immune suppression. Thus, SCD1 inhibitors should increase anti-tumor immunity. We evaluated the effect of SSI4-mediated SCD1 inhibition in two mouse triple negative breast cancer cell lines. The data showed that SSI4 stimulates calreticulin (CRT) translocation to cell membrane. CRT is an ER resident protein that translocates to the cell surface under ER stress conditions. Cell surface expression of CRT is known to induce immune responses including phagocytosis, translocation of cancer antigen to the cell surface, and apoptosis. We are currently studying the effect of CRT translocation in our model. A major focus of our project is to assess the efficacy of combination treatment of SSI4 and an immune checkpoint blockade. To this end, this combination therapy synergistically blocked E0771 tumor growth in vivo. Further, to understand the immune responses generated, we performed immunohistochemical analysis of the tumors (day 20 post-treatment) indicating that the combination therapy resulted in higher membrane localized CRT, supporting our rationale that CRT translocation is involved in mediating the anti-tumor responses. Flow cytometry analysis indicated that the combination treatment increased immune cell infiltration. Specifically, SSI4 treatment induced infiltration of double negative T cells, natural killer cells and macrophages, suggesting that it promoted a pro-inflammatory immune response. SSI4 treatment down regulated immune suppressive cells including Tregs, dendritic cells and mesenchymal derived stem cells. The response of cancers with low and intermediate tumor mutational burden such as breast cancer to immune checkpoint inhibition is limited. Our data indicates that combination treatment of SSI4 and anti PD1 is a promising therapeutic strategy. Citation Format: Sneha Vivekanandhan, Justyna Trynda, Laura A. Marlow, Barath shreeder, James L. Miller, Adam M. Kase, Winston Tan, Keith L. Knutson, John A. Copland. SCD-1 blockade sensitizes triple negative breast cancer to immune checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1484.

Longitudinal changes in fatty acid metabolism and in the mitochondrial protein import system in overconditioned and normal conditioned cows: A transcriptional study using microfluidic quantitative PCR

This study investigated the effect of body condition around calving on the hepatic mRNA expression of genes involved in fatty acid (FA) metabolism and mitochondrial protein import system of dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets to reach targeted differences in BCS and backfat thickness (BFT) until dry-off. At dry-off, normal BCS (NBCS) cows had a BCS <3.5 and BFT <1.2 cm, and the high BCS (HBCS) cows had a BCS >3.75 and BFT >1.4 cm. The expression of targeted genes in the liver was assayed by reverse-transcription quantitative real-time PCR using microfluidics integrated fluidic circuit chips on a subset of 5 cows from each group. Liver biopsies were collected at d -49, +3, +21, and +84 relative to parturition. The mRNA abundance of 47 genes related to lipid metabolism including carnitine metabolism, FA uptake and transport, lipoprotein export, carnitine metabolism, mitochondrial and proximal FA oxidation, ketogenesis, AMP-activated protein kinase/mammalian target of rapamycin pathway, and mitochondrial protein import system was assessed in liver tissue. The mRNA abundances of FA binding protein (FABP)6 (in both groups), and FABP1 and solute carrier family 22 member 5 (SLC22A5) in HBCS were upregulated (>1.5-fold change, FC) in early lactation (at d +3 and +21 postpartum) compared with antepartum (d -49), indicating promoted FA uptake and intracellular transport in the liver due to the metabolic adaptations of elevated lipo-mobilization after parturition. The upregulation of SLC22A5 and SLC25A20 after parturition was more pronounced in HBCS than in NBCS cows, suggesting a need for increasing the capacity of FA uptake, and FA transport into the hepatocyte. The increased mRNA abundance of carnitine palmitoyltransferase 1A, after parturition and to a greater extent in HBCS (FC = 4.1) versus NBCS (FC = 2.1) indicates a physiological increase in the capacity of long-chain fatty acyl-CoA entry into the liver mitochondria compared with antepartum (ap; d -49 relative to calving). The greater hepatic mRNA abundance of genes encoding enzymes involved in mitochondrial FA oxidation in HBCS than in NBCS points to an increased rate of mitochondrial β-oxidation. The hepatic mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 2 and 3-hydroxy-3-methylglutaryl-CoA were upregulated after parturition (d +21/d +3 pp) to a greater extent in HBCS than in NBCS cows, indicating that excess acetyl-CoA generated via β-oxidation was increasingly used for ketogenesis. We observed for the first time that the mRNA abundance of genes involved in the translocase of the inner membrane (TIM) complex (TIM22 and TIM23) in the hepatic mitochondrial protein import system were undergoing distinct changes during the transition from late pregnancy to early lactation in dairy cows. Even though sample size in this study was relatively small, the results support that overconditioning around calving may contribute to mitochondrial FA overload and greater ketogenesis at the level of transcription in the liver of early lactation cows.

Biochemical Characterization of Acyl-CoA: Lysophosphatidylcholine Acyltransferase (LPCAT) Enzyme from the Seeds of Salvia hispanica.

Salvia hispanica (chia) is the highest reported terrestrial plant source of alpha-linolenic acid (ALA, ~ 65%), an ω-3 polyunsaturated fatty acid with numerous health benefits. The molecular basis of high ALA accumulation in chia is yet to be understood. We have identified lysophosphatidylcholine acyltransferase (LPCAT) gene from the developing seed transcriptome data of chia and carried out its biochemical characterization through heterologous expression in Saccharomyces cerevisiae. Expression profiling showed that the enzyme was active throughout the seed development, indicating a pivotal role in oil biosynthesis. The enzyme could utilize both saturated and unsaturated lysophosphatidylcholine substrates at the same rate, to synthesize phosphatidylcholine (PC). The enzyme also exhibited lysophosphatidic acid acyltransferase (LPAAT) activity, by preferring lysophosphatidic acid substrate. Substrate specificity studies showed that the enzyme preferred both monounsaturated and polyunsaturated fatty acyl CoAs over saturated CoAs. This activity may play a key role in enriching the PC fraction with polyunsaturated fatty acids (PUFAs). PUFAs present on PC can be transferred to oil through the action of other acyltransferases. Our results describe a new LPCAT enzyme that can be used to biotechnologically alter oilseed crops to incorporate more PUFA in its seed oil.

Thioesterase superfamily member 1 undergoes stimulus-coupled conformational reorganization to regulate metabolism in mice

In brown adipose tissue, thermogenesis is suppressed by thioesterase superfamily member 1 (Them1), a long chain fatty acyl-CoA thioesterase. Them1 is highly upregulated by cold ambient temperature, where it reduces fatty acid availability and limits thermogenesis. Here, we show that Them1 regulates metabolism by undergoing conformational changes in response to β-adrenergic stimulation that alter Them1 intracellular distribution. Them1 forms metabolically active puncta near lipid droplets and mitochondria. Upon stimulation, Them1 is phosphorylated at the N-terminus, inhibiting puncta formation and activity and resulting in a diffuse intracellular localization. We show by correlative light and electron microscopy that Them1 puncta are biomolecular condensates that are inhibited by phosphorylation. Thus, Them1 forms intracellular biomolecular condensates that limit fatty acid oxidation and suppress thermogenesis. During a period of energy demand, the condensates are disrupted by phosphorylation to allow for maximal thermogenesis. The stimulus-coupled reorganization of Them1 provides fine-tuning of thermogenesis and energy expenditure.

The Effect of L- Carnitine on Oocyte Maturity in Women with Polycystic Ovarian Syndrome undergone ICSI

Due to malfunction of the hypothalamus-pituitary-ovary axis and probable dysregulation of ovarian activities such as follicle selection or follicular atresia, PCOS patients' ovary has a large amount of small follicles but no developed follicles. L-carnitine is a tiny, water-soluble chemical that plays a critical role in fat metabolism. It is required for appropriate mitochondrial fatty acid oxidation and acyl-CoA ester excretion, as well as affecting ATP levels. The study was conducted in High Institute of Infertility Diagnosis and Assisted Reproductive Technologies, Al Nahrain University and Al-Farah private I.V.F center, 60 infertile women diagnosed with polycystic ovarian syndrome have been enrolled in this study and enter their IVF/ICSI cycle; they were informed about the study and signed a written informed consent. They were grouped into treatment group (n=30) who were treated with L-Carnitine® tablet for two months (VERO UNIVERSAL and NOW) and placebo group (n=30). All enrolled women underwent flexible antagonist protocol for ICSI. Significant increase in mean mature metaphase II (MII) oocyte count in study group than placebo group, 9.17 ±2.63 versus 7.27 ±3.49, respectively (p=0.021), oocyte maturity rate was significantly higher in study group in comparison with placebo group, 84.52 ±13.65 % versus 62.30 ±16.56 %, respectively (p &lt; 0.001). In addition, mean fertilized (2PN) oocyte count was increased significantly in study group in comparison with placebo group, 7.93 ±2.63 versus 6.77 ±3.07, respectively (p=0.042). Mean fertilization rate was significantly higher in study group in comparison with placebo group, 66.79 ±14.05 % versus 56.67 ±19.05 %, respectively (p = 0.035).

The Effect of L- Carnitine on Oocyte Maturity in Women with Polycystic Ovarian Syndrome Undergoing ICSI

Due to malfunction of the hypothalamus-pituitary-ovary axis and probable dysregulation of ovarian activities such as follicle selection or follicular atresia, PCOS patients' ovary has a large amount of small follicles but no developed follicles. L-carnitine is a tiny, water-soluble chemical that plays a critical role in fat metabolism. It is required for appropriate mitochondrial fatty acid oxidation and acyl-CoA ester excretion, as well as affecting ATP levels. The study was conducted in High Institute of Infertility Diagnosis and Assisted Reproductive Technologies, Al Nahrain University and Al-Farah private I.V.F center, 60 infertile women diagnosed with polycystic ovarian syndrome have been enrolled in this study and enter their IVF/ICSI cycle; they were informed about the study and signed a written informed consent. They were grouped into treatment group (n=30) who were treated with L-Carnitine® tablet for two months (VERO UNIVERSAL and NOW) and placebo group (n=30). All enrolled women underwent flexible antagonist protocol for ICSI. Significant increase in mean mature metaphase II (MII) oocyte count in study group than placebo group, 9.17 ±2.63 versus 7.27 ±3.49, respectively (p=0.021), oocyte maturity rate was significantly higher in study group in comparison with placebo group, 84.52 ±13.65 % versus 62.30 ±16.56 %, respectively (p &lt; 0.001). In addition, mean fertilized (2PN) oocyte count was increased significantly in study group in comparison with placebo group, 7.93 ±2.63 versus 6.77 ±3.07, respectively (p=0.042). Mean fertilization rate was significantly higher in study group in comparison with placebo group, 66.79 ±14.05 % versus 56.67 ±19.05 %, respectively (p = 0.035).

31-OR: ACSL1 in Smooth Muscle Cells Regulates High-Fat Diet-Induced Obesity and Thermogenesis in Mice

Long-chain acyl-CoA synthetases (ACSL) are the enzymes that convert long-chain fatty acids to long-chain fatty acyl-CoAs that are utilized for various cellular processes, including triglyceride synthesis, energy metabolism, sphingolipid formation, and modification of lipids and proteins. Among five common isotypes (ACSL1, 3, 4, 5, and 6), ACSL1 is the predominant isotype in adipose tissue and essential for fatty acid oxidation during the cold adaption in brown adipose tissue. However, it is unknown whether ACSL1 is involved in brown adipocyte differentiation. Since the smooth muscle-like cells (mural cells) are the progenitors of brown adipocytes, we hypothesize that the ACSL1 in smooth muscle cells contributes to brown adipocyte differentiation and energy metabolism. We generated a smooth muscle-specific ACSL1 knockout mouse (ACSL1SM-/-) by breeding SM22α-cre mouse with ACSL1flox/flox mouse. ACSL1 deficiency in smooth muscle cells protected from high-fat diet (HFD)-induced obesity without changes in food intake and physical activity. Furthermore, HFD-fed ACSL1flox/flox mice were more glucose tolerant and insulin sensitive compared to HFD-fed wild-type mice. We also observed that cold adaptation, thermogenesis, was significantly impaired in ACSL1SM-/- mice. We isolated stromal vascular cells (SVCs) from inguinal and brown adipose tissues and then subjected them to adipocyte differentiation. We found that the adipocyte differentiation was impaired, and the expression of UCP1 was significantly reduced in the absence of ACSL1. These results suggest that ACSL1 in smooth muscle cells contributes to energy metabolism and glucose homeostasis by regulation of brown fat differentiation. Disclosure G. Ren: None. M. Young: None. J. Kim: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK079626); National Heart, Lung, and Blood Institute (HL128695); National Institute on Aging (AG058078)

Comparative analysis of alfalfa (Medicago sativa L.) seedling transcriptomes reveals genotype-specific drought tolerance mechanisms

Drought is one of the main abiotic factors that affect alfalfa yield. The identification of genes that control this complex trait can provide important insights for alfalfa breeding. However, little is known about how alfalfa responds and adapts to drought stress, particularly in cultivars of differing drought tolerance. In this study, the drought-tolerant cultivar Dryland ‘DT’ and the drought-sensitive cultivar WL343HQ ‘DS’ were used to characterize leaf and root physiological responses and transcriptional changes in response to water deficit. Under drought stress, Dryland roots (DTR) showed more differentially expressed genes than WL343HQ roots (DSR), whereas WL343HQ leaves (DSL) showed more differentially expressed genes than Dryland leaves (DTL). Many of these genes were involved in stress-related pathways, carbohydrate metabolism, and lignin and wax biosynthesis, which may have improved the drought tolerance of alfalfa. We also observed that several genes related to ABA metabolism, root elongation, peroxidase activity, cell membrane stability, ubiquitination, and genetic processing responded to drought stress in alfalfa. We highlighted several candidate genes, including sucrose synthase, xylan 1,4-beta-xylosidase, primary-amine oxidase, and alcohol-forming fatty acyl-CoA reductase, for future studies on drought stress resistance in alfalfa and other plant species. In summary, our results reveal the unique drought adaptation and resistance characteristics of two alfalfa genotypes. These findings, which may be valuable for drought resistance breeding, warrant further gene functional analysis to augment currently available information and to clarify the drought stress regulatory mechanisms of alfalfa and other plants.

Peripheral Insulin Regulates a Broad Network of Gene Expression in Hypothalamus, Hippocampus, and Nucleus Accumbens.

The brain is now recognized as an insulin-sensitive tissue; however, the role of changing insulin concentrations in the peripheral circulation in gene expression in the brain is largely unknown. Here, we performed a hyperinsulinemic-euglycemic clamp on 3-month-old male C57BL/6 mice for 3 h. We show that, in comparison with results in saline-infused controls, increases in peripheral insulin within the physiological range regulate expression of a broad network of genes in the brain. Insulin regulates distinct pathways in the hypothalamus (HTM), hippocampus, and nucleus accumbens. Insulin shows its most robust effect in the HTM and regulates multiple genes involved in neurotransmission, including upregulating expression of multiple subunits of GABA-A receptors, Na+ and K+ channels, and SNARE proteins; differentially modulating glutamate receptors; and suppressing multiple neuropeptides. Insulin also strongly modulates metabolic genes in the HTM, suppressing genes in the glycolysis and pentose phosphate pathways, while increasing expression of genes regulating pyruvate dehydrogenase and long-chain fatty acyl-CoA and cholesterol biosynthesis, thereby rerouting of carbon substrates from glucose metabolism to lipid metabolism required for the biogenesis of membranes for neuronal and glial function and synaptic remodeling. Furthermore, based on the transcriptional signatures, these changes in gene expression involve neurons, astrocytes, oligodendrocytes, microglia, and endothelial cells. Thus, peripheral insulin acutely and potently regulates expression of a broad network of genes involved in neurotransmission and brain metabolism. Dysregulation of these pathways could have dramatic effects in normal physiology and diabetes.

Expression Profile of Acetyl CoA Carboxylase Beta (ACACB) Gene during the Pre and Post-hatch Period in Chicken

Background: Acetyl-CoA Carboxylase Beta (ACACB) plays a key role in fatty acid oxidation and was known to be involved in production of very-long-chain fatty acid and other compounds needed for proper development. This gene is mainly expressed in the tissues of heart, muscle, liver and colon. It chiefly involved in the production of malonyl-coA, a potent inhibitor of carnitine palmitoyl transferase I (CPT-I) enzyme needed in transport of long-chain fatty acyl-coAs to the mitochondria for β-oxidation.Methods: The present study was conducted to explore the expression pattern of the ACACB gene in breast muscle tissue during pre-hatch embryonic day (ED) 5th to 18th and post-hatch (18th, 22nd and 40th week of age) periods of White leghorn (IWI line) by using Quantitative real-time PCR (qPCR). Then, fold change of ACACB gene expression was calculated.Result: Our study showed that the ACACB gene expression was down-regulated during embryonic stages from ED6 to ED18. The gene expression was also down-regulated during adult stages i.e. on 22nd and 40th week of age. This result indicated that the initial expression of the ACACB gene is required for embryo development and during adult periods, low gene expression leads to the less fat deposition in muscle of layer chicken. Finally, it can be concluded that there was a differential expression pattern of the ACACB gene during the pre-hatch embryonic and post-hatch adult periods to mitigate varied requirements of lipids during different physiological stages in layer chicken.

ATPase Effects on Pro-Nutrients-mTOR Release Long Fatty Acids Chains which Under Mitochondrial Phospholipase, Synthase, &amp; Synthetase Effects form Three ROR-Alpha, Beta, Gamma Isoforms

ATPase Effects on Pro-Nutrients-mTOR Release Long Fatty Acids Chains which Under Mitochondrial Phospholipase, Synthase, &amp; Synthetase Effects form Three ROR-Alpha, Beta, Gamma Isoforms

Targeting Them1 for the Management of Obesity‐Related Disorders

Background Thioesterase superfamily member 1 (Them1; synonyms acyl-CoA thioesterase 11 and StarD14) is a fatty acyl-CoA thioesterase that hydrolyzes long-chain fatty acyl-CoAs into free fatty acids plus CoASH. Genetic disruption of Them1 in mice leads to increased energy expenditure, along with resistance to high fat diet-induced obesity, diabetes and non-alcoholic fatty liver disease. Them1 is comprised of a N-terminal enzymatic domain linked to a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain that can allosterically modulate enzymatic activity. Taken together, these findings suggest that chemical inhibition of Them1 could be leveraged in the management of obesity-related disorders. Aim This study was designed to develop a small molecule inhibitor that targets Them1 activity. Methods Recombinant Them1 protein was expressed in E. coli and purified using a N-terminal His-tag. A fluorescence-based assay to detect free CoASH liberated by the activity of Them1 was optimized and miniaturized into 384-well plates. A high-throughput screen utilizing a highly diverse small molecule library (360,705 compounds) was conducted to identify small molecule inhibitors targeting Them1 activity. The threshold for small molecule inhibitors was set to normalized percent inhibition > 30 and z-score < -3. To select Them1-specific small molecule inhibitors, a counter screen was conducted targeting other Acot isoforms, as well as a truncated Them1 containing only the enzymatic domain (i.e. lacking the START domain). To further improve the potency of small molecules to inhibit Them1 activity, initial structure activity relationship (SAR) expansion was performed. Biomolecular microscale thermophoresis assays were utilized to determine binding affinities of small molecule inhibitors to Them1. Results 330 small molecules were identified from the high-throughput screen (hit rate 0.09%). Ten small molecules derived from the counter screen showed specificity toward inhibiting Them1 activity either through the enzymatic (3 compounds; IC50: 4 – 10 μM) or START (7 compounds; IC50: 6 – 20 μM) domains. SAR analyses revealed molecular features that influenced the IC50 values for selected small molecules. Three small molecules exhibited Kd values in the low μM range. Conclusions We have identified small molecule inhibitors targeting Them1 activity. An optimized small molecule inhibitor targeting Them1 activity should become an attractive candidate for the pharmacologic management of obesity-related disorders.

Study of The Divergent Acyl Chain Specificities of The Class I Ceramide Synthases in Plants

Ceramide synthases catalyze the condensation of long-chain bases and fatty acyl-CoA to form the ceramide backbones of glycosphingolipids.The Class I and II ceramide synthases of plants have distinct substrate specificities that contribute to the divergent fatty acid chain-lengths and functions of plant sphingolipids. The Arabidopsis LOH2-encoded Class I ceramide synthase has been shown to have strong specificity for palmitoyl (16:0)-CoA that gives rise to ceramides with C16 fatty acids that are prevalent in dicot glucosylceramides. By contrast, glucosylceramides of many monocots and selected dicots are enriched in C18 and C20-ceramides. We hypothesized that these differences are due to functional and structural differences in LOH2 ceramide synthases. Consistent with this, we found that transgenic expression of a tomato LOH2 restored C16 fatty acid in glucosylceramides in an Arabidopsis loh2 mutant, whereas expression of a rice LOH2 resulted in C18- and C20-fatty acid-enriched glucosylceramides. To establish the structural basis for these different outcomes, we demonstrated that swapping a divergent 16 amino acid domain from the rice LOH2 to tomato LOH2 yielded C18- and C20-fatty acid-glucosylceramides when expressed in the loh2 mutant. By comparison of primary structures of LOH2 enzymes from diverse dicots and monocots, we identified a single amino acid that when mutated in the tomato LOH2 resulted in rice-type C18- and C20 fatty acid-glucosylceramides.We rationalize the altered LOH2 function in the context of the predicted LOH2 ceramide synthase secondary structure. Overall, our findings highlight the biochemical and genetic basis for divergent ceramide structures in dicots and monocots.

A Functional mTORC1 Containing Raptor Controls Luminal Fat Processing and Epithelium Barrier Function in Transwell Caco‐2 Cultures

As a critical regulatory point of nutrient sensing, growth and metabolism, the mechanistic target of rapamycin complex 1 (mTORC1) is poised to influence intestinal homeostasis under basal conditions and in disease state. We created three stable human colorectal adenocarcinoma Caco-2 cell lines exhibiting normal, low (Raptor knockdown) or high (tuberin knockdown) mTORC1 kinase activity, and grew these cells in Transwell to investigate the contribution of mTORC1 in fat absorption, processing, and barrier permeability. Raptor knockdown impaired lipid and apoB-containing lipoprotein metabolism in Caco-2 cultures provided exogenous oleate in the apical chamber. Raptor-depleted Caco-2 cells secreted less apoB100 (-51%), apoB48 (-66%) and triacylglycerol (-69%) compared with control Caco-2 cells. Genes involved in lipogenesis (FASN, fatty acid synthase), fatty acyl-CoA formation (FATP2, very long-chain acyl-CoA ligase; DBI, diazepam binding inhibitor acyl-CoA binding protein), triacylglycerol synthesis (DGAT1, diacylglycerol O-acyltransferase 1; DGAT2, diacylglycerol O-acyltransferase 2), lipoprotein assembly (APOB, apolipoprotein B; MTTP, microsomal triglyceride transfer protein), and pre-chylomicron transport (VAMP7,vesicle-associated membrane protein 7; SAR1B, secretion associated Ras related GTPase 1B) were downregulated by at least 60% in Caco-2 cells depleted of Raptor versus control Caco-2 cells. The cellular protein abundance of apoB (-89%), MTTP (-63%) and FASN (-80%) were decreased in Raptor-depleted versus control Caco-2 cells. When mTORC1 activity was abrogated, Transwell-type Caco-2 cultures were impermeable to ions (high transepithelial electrical resistance, TEER) and had elevated paracellular transport of 4-kDa FITC dextran. Raptor knockdown likewise rapamycin, a mTORC1 inhibitor, decreased the abundance of small-sized solute pore-forming claudins 2 and 15, and the levels of tight junction proteins occludin and tricellulin. In contrast, resistance against ion diffusion decreased (low TEER) and paracellular transport of 4-kDa FITC dextran increased as mTORC1 activity rose. In conclusion, a functional intestinal mTORC1 is critically important for processing dietary lipids into chylomicrons and establishing the permeability characteristics of the epithelium.

Solution and Membrane Interaction Dynamics of Mycobacterium tuberculosis Fatty Acyl-CoA Synthetase FadD13

The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism. FadD13 is a peripheral membrane protein, with both soluble and membrane-bound populations in vivo. The protein displays a distinct positively charged surface patch, suggested to be involved in membrane association. In this paper, we combine structural analysis with liposome co-flotation assays and membrane association modeling to gain a more comprehensive understanding of the mechanisms behind membrane association. We show that FadD13 has affinity for negatively charged lipids, such as cardiolipin. Addition of a fatty acid substrate to the liposomes increases the apparent affinity of FadD13, consistent with our previous hypothesis that FadD13 can utilize the membrane to harbor its very-long-chain fatty acyl substrates. In addition, we unambiguously show that FadD13 adopts a dimeric arrangement in solution. The dimer interface partly buries the positive surface patch, seemingly inconsistent with membrane binding. Notably, when cross-linking the dimer, it lost its ability to bind and co-migrate with liposomes. To better understand the dynamics of association, we utilized two mutant variants of FadD13, one in which the positively charged patch was altered to become more negative and one more hydrophobic. Both variants were predominantly monomeric in solution. The hydrophobic variant maintained the ability to bind to the membrane, whereas the negative variant did not. Taken together, our data indicate that FadD13 exists in a dynamic equilibrium between the dimer and monomer, where the monomeric state can adhere to the membrane via the positively charged surface patch.

BdFAR4, a root-specific fatty acyl-coenzyme A reductase, is involved in fatty alcohol synthesis of root suberin polyester in Brachypodium distachyon.

Suberin is a complex hydrophobic polymer of aliphatic and phenolic compounds which controls the movement of gases, water, and solutes and protects plants from environmental stresses and pathogenic infection. The synthesis and regulatory pathways of suberin remain unknown in Brachypodium distachyon. Here we describe the identification of a B. distachyon gene, BdFAR4, encoding a fatty acyl-coenzyme A reductase (FAR) by a reverse genetic approach, and investigate the molecular relevance of BdFAR4 in the root suberin synthesis of B. distachyon. BdFAR4 is specifically expressed throughout root development. Heterologous expression of BdFAR4 in yeast (Saccharomyces cerevisiae) afforded the production of C20:0 and C22:0 fatty alcohols. The loss-of-function knockout of BdFAR4 by CRISPR/Cas9-mediated gene editing significantly reduced the content of C20:0 and C22:0 fatty alcohols associated with root suberin. In contrast, overexpression of BdFAR4 in B. distachyon and tomato (Solanum lycopersicum) resulted in the accumulation of root suberin-associated C20:0 and C22:0 fatty alcohols, suggesting that BdFAR4 preferentially accepts C20:0 and C22:0 fatty acyl-CoAs as substrates. The BdFAR4 protein was localized to the endoplasmic reticulum in Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaf epidermal cells. BdFAR4 transcript levels can be increased by abiotic stresses and abscisic acid treatment. Furthermore, yeast one-hybrid, dual-luciferase activity, and electrophoretic mobility shift assays indicated that the R2R3-MYB transcription factor BdMYB41 directly binds to the promoter of BdFAR4. Taken together, these results imply that BdFAR4 is essential for the production of root suberin-associated fatty alcohols, especially under stress conditions, and that its activity is transcriptionally regulated by the BdMYB41 transcription factor.

Acyl-CoA desaturase ADS4.2 is involved in the formation of characteristic wax alkenes in young Arabidopsis leaves.

Monounsaturated alkenes are present in the cuticular waxes of diverse plants and are thought to play important roles in their interactions with abiotic and biotic factors. Arabidopsis (Arabidopsis thaliana) leaf wax has been reported to contain alkenes; however, their biosynthesis has not been investigated to date. Here, we found that these alkenes have mainly ω-7 and ω-9 double bonds in characteristically long hydrocarbon chains ranging from C33 to C37. A screening of desaturase-deficient mutants showed that a single desaturase belonging to the acyl-CoA desaturase (ADS) family, previously reported as ADS4.2, was responsible for introducing double bonds en route to the wax alkenes. ADS4.2 was highly expressed in young leaves, especially in trichomes, where the alkenes are known to accumulate. The enzyme showed strong activity on acyl substrates longer than C32 and ω-7 product regio-specificity when expressed in yeast (Saccharomyces cerevisiae). Its endoplasmic reticulum localization further confirmed that ADS4.2 has access to very-long-chain fatty acyl-CoA substrates. The upstream biosynthesis pathways providing substrates to ADS4.2 and the downstream reactions forming the alkene products in Arabidopsis were further clarified by alkene analysis of mutants deficient in other wax biosynthesis genes. Overall, our results show that Arabidopsis produces wax alkenes through a unique elongation-desaturation pathway, which requires the participation of ADS4.2.

Progesterone receptor membrane component 1 reduces cardiac steatosis and lipotoxicity via activation of fatty acid oxidation and mitochondrial respiration

Obesity is implicated in cardiovascular disease and heart failure. When fatty acids are transported to and not adequately oxidized in cardiac cells, they accumulate, causing lipotoxicity in the heart. Since hepatic progesterone receptor membrane component 1 (Pgrmc1) suppressed de novo lipogenesis in a previous study, it was questioned whether cardiac Pgrmc1 protects against lipotoxicity. Hence, we focused on the role of cardiac Pgrmc1 in basal (Resting), glucose-dominant (Refed) and lipid-dominant high-fat diet (HFD) conditions. Pgrmc1 KO mice showed high FFA levels and low glucose levels compared to wild-type (WT) mice. Pgrmc1 KO mice presented low number of mitochondrial DNA copies in heart, and it was concomitantly observed with low expression of TCA cycle genes and oxidative phosphorylation genes. Pgrmc1 absence in heart presented low fatty acid oxidation activity in all conditions, but the production of acetyl-CoA and ATP was in pronounced suppression only in HFD condition. Furthermore, HFD Pgrmc1 KO mice resulted in high cardiac fatty acyl-CoA levels and TG level. Accordingly, HFD Pgrmc1 KO mice were prone to cardiac lipotoxicity, featuring high levels in markers of inflammation, endoplasmic reticulum stress, oxidative stress, fibrosis, and heart failure. In vitro study, it was also confirmed that Pgrmc1 enhances rates of mitochondrial respiration and fatty acid oxidation. This study is clinically important because mitochondrial defects in Pgrmc1 KO mice hearts represent the late phase of cardiac failure.

Enhancement of C6–C10 fatty acid ethyl esters production in Saccharomyces cerevisiae CA by metabolic engineering

C6–C10 fatty acid ethyl esters (FAEEs) are important aromatic substances in alcoholic beverages and include ethyl hexanoate (EH), ethyl octanoate (EO), and ethyl decanoate (ED). However, the biosynthesis capacity of these esters are limited because of poor precursor C6–C10 fatty acyl-CoA accumulation and low alcohol acyltransferase (AATase) activity levels in Saccharomyces cerevisiae. In this study, S. cerevisiae CA stain was selected as the host stain because of its relatively high C6–C10 FAEE productivity. Ester production was improved by metabolic engineering strategies. Acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (ACS1) were overexpressed to enhance the acetyl-CoA precursor accumulation. Intracellular malonyl-CoA availability was also improved by acetyl-CoA decarboxylase (ACC1Ser659Ala, Ser1157Ala) overexpression. FAS1 and FAS2 (encoding fatty acid synthase) overexpression facilitated the condensation reactions to form C6–C10 fatty acyl-CoA from malonyl-CoA and acetyl-CoA precursors. Furthermore, the strawberry alcohol acyltransferase (SAAT) was introduced into S. cerevisiae CA to catalyze the formation of C6–C10 FAEEs from C6–C10 fatty acyl-CoA and ethanol substrates. As a consequence, the newly engineered strain achieved 25.89-, 7.27-, and 9.05-fold increases in extracellular EH (7.53 mg/L), EO (13.65 mg/L) and ED (13.87 mg/L) levels, respectively, compared to the wild-type strain CA.

Nutrient infusion in the dorsal vagal complex controls hepatic lipid and glucose metabolism in rats

SummaryHypothalamic regulation of lipid and glucose homeostasis is emerging, but whether the dorsal vagal complex (DVC) senses nutrients and regulates hepatic nutrient metabolism remains unclear. Here, we found in rats DVC oleic acid infusion suppressed hepatic secretion of triglyceride-rich very-low-density lipoprotein (VLDL-TG), which was disrupted by inhibiting DVC long-chain fatty acyl-CoA synthetase that in parallel disturbed lipid homeostasis during intravenous lipid infusion. DVC glucose infusion elevated local glucose levels similarly as intravenous glucose infusion and suppressed hepatic glucose production. This was independent of lactate metabolism as inhibiting lactate dehydrogenase failed to disrupt glucose sensing and neither could DVC lactate infusion recapitulate glucose effect. DVC oleic acid and glucose infusion failed to lower VLDL-TG secretion and glucose production in high-fat fed rats, while inhibiting DVC farnesoid X receptor enhanced oleic acid but not glucose sensing. Thus, an impairment of DVC nutrient sensing may lead to the disruption of lipid and glucose homeostasis in metabolic syndrome.